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The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex.

Abdul Azeez KR, Knapp S, Fernandes JM, Klussmann E, Elkins JM - Biochem. J. (2014)

Bottom Line: The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF.Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA.Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

View Article: PubMed Central - PubMed

Affiliation: *Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.

ABSTRACT
The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA-GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)-PH (pleckstrin homology)] domain to 2.1 Å (1 Å = 0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA-AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

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Structure of AKAP-Lbc(A) Protein domains of AKAP-Lbc (AKAP13). The range of the construct used for structure determination is shown above the DH and PH domains. (B) Overview of the complex between RhoA–GDP and the DH–PH domains of AKAP-Lbc. RhoA is shown with red α-helices and yellow β-strands, and with the switch I and switch II loops in green. AKAP-Lbc is shown with its DH domain in blue and PH domain in green. (C) The binding of the RhoA switch I loop. The switch I loop is shown in yellow, with the AKAP-Lbc DH domain shown in green. (D) The binding of the RhoA switch II loop, coloured as for (C).
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Figure 1: Structure of AKAP-Lbc(A) Protein domains of AKAP-Lbc (AKAP13). The range of the construct used for structure determination is shown above the DH and PH domains. (B) Overview of the complex between RhoA–GDP and the DH–PH domains of AKAP-Lbc. RhoA is shown with red α-helices and yellow β-strands, and with the switch I and switch II loops in green. AKAP-Lbc is shown with its DH domain in blue and PH domain in green. (C) The binding of the RhoA switch I loop. The switch I loop is shown in yellow, with the AKAP-Lbc DH domain shown in green. (D) The binding of the RhoA switch II loop, coloured as for (C).

Mentions: AKAP-Lbc has a RhoGEF DH–PH domain between residues 1972 and 2342 (Figure 1A). Its PH domain was shown to be dispensable for activation of Rho, but important for the localization and transformative activity of AKAP-Lbc [25]. The region C-terminal to the DH–PH domain is involved in scaffolding PRKCH [also known as PKCη (protein kinase Cη)] and PRKD1 (protein kinase D1) to activate PRKD1 [8]. This C-terminal region is altered in oncogenic AKAP-Lbc [26].


The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex.

Abdul Azeez KR, Knapp S, Fernandes JM, Klussmann E, Elkins JM - Biochem. J. (2014)

Structure of AKAP-Lbc(A) Protein domains of AKAP-Lbc (AKAP13). The range of the construct used for structure determination is shown above the DH and PH domains. (B) Overview of the complex between RhoA–GDP and the DH–PH domains of AKAP-Lbc. RhoA is shown with red α-helices and yellow β-strands, and with the switch I and switch II loops in green. AKAP-Lbc is shown with its DH domain in blue and PH domain in green. (C) The binding of the RhoA switch I loop. The switch I loop is shown in yellow, with the AKAP-Lbc DH domain shown in green. (D) The binding of the RhoA switch II loop, coloured as for (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232260&req=5

Figure 1: Structure of AKAP-Lbc(A) Protein domains of AKAP-Lbc (AKAP13). The range of the construct used for structure determination is shown above the DH and PH domains. (B) Overview of the complex between RhoA–GDP and the DH–PH domains of AKAP-Lbc. RhoA is shown with red α-helices and yellow β-strands, and with the switch I and switch II loops in green. AKAP-Lbc is shown with its DH domain in blue and PH domain in green. (C) The binding of the RhoA switch I loop. The switch I loop is shown in yellow, with the AKAP-Lbc DH domain shown in green. (D) The binding of the RhoA switch II loop, coloured as for (C).
Mentions: AKAP-Lbc has a RhoGEF DH–PH domain between residues 1972 and 2342 (Figure 1A). Its PH domain was shown to be dispensable for activation of Rho, but important for the localization and transformative activity of AKAP-Lbc [25]. The region C-terminal to the DH–PH domain is involved in scaffolding PRKCH [also known as PKCη (protein kinase Cη)] and PRKD1 (protein kinase D1) to activate PRKD1 [8]. This C-terminal region is altered in oncogenic AKAP-Lbc [26].

Bottom Line: The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF.Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA.Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

View Article: PubMed Central - PubMed

Affiliation: *Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.

ABSTRACT
The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA-GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)-PH (pleckstrin homology)] domain to 2.1 Å (1 Å = 0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA-AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

Show MeSH
Related in: MedlinePlus